Method and device for scanning wells in a multi-well plate

ABSTRACT

An auto-focusing method for determining an in-focus position of a plurality of wells in at least a portion of a multi-well plate, the method including using a first objective lens having a first magnification to identify, in each of at least three wells of a selected subset of the plurality of wells, an in-focus position of each well with respect to the first objective lens, on the basis of at least three the in-focus positions, computing a plane along which the at least three wells will be in focus with respect to at least one objective lens having a second magnification that is not greater than the first magnification, and using the at least one objective lens to scan, along the plane, at least some of the plurality of wells in the portion of the plate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/IB2016/050323, filedJan. 22, 2016 and entitled “Auto-Focusing Method And Device”, whichclaims priority from GB 1501093.7, filed Jan. 22, 2015. The benefitand/or priority, as appropriate, of both of these applications isclaimed, and the contents of both of said applications are incorporatedherein by reference.

FIELD AND BACKGROUND

The present invention generally relates to the field of opticalmeasurement and/or inspection techniques and more specifically relatesto an auto-focus method and device, particularly useful when viewing nonplanar surfaces.

Auto focusing is an essential feature in many automated inspectionfields such as the computer chip industry, biomedical research, datareading/recording in optical information carriers, etc. Specifically,when analyzing samples in multiwell plates including a plurality ofwells on a single plate, auto focusing of a microscope viewing thecontents of the wells can enable more efficient work procedures as theoperator need not focus the objective on each well in the plateseparately.

Various auto focusing methods for inspection of a multiwell plate havebeen disclosed in the past, such as in U.S. Pat. No. 7,109,459. However,when using wells having a non-planar bottom, such as a multiwell platehaving wells with a U-shaped bottom, such as are used, for example, forgrowing living cells into spheroids, existing autofocus methods mayrequire image analysis which is time-consuming.

In some cases, a multiwell plate is analyzed or is visualized using anoil immersion lens having a high numerical aperture. In such cases,focusing the objective using laser based autofocusing methods asdescribed in U.S. Pat. No. 7,109,459 is difficult or impossible, as thesimilar refraction index of the lens and solution in the wells cancelsone of the refraction points and a corresponding scanning peak, causingdifficulty in focusing the objective lens.

Thus there are needs: (a) for auto focusing a microscope on a multiwellplate, which is suitable for multi-well plates having wells with anon-planar bottom surface; (b) to separate focusing process fromscanning process to allow higher scanning and imaging speed; and (c) fora method for auto focusing a microscope using an oil immersion lens on amultiwell plate.

SUMMARY

The present invention generally relates to the field of opticalmeasurement and/or inspection techniques and more specifically relatesto an auto-focus method and device, particularly useful when viewing nonplanar surfaces.

There is provided in accordance with an embodiment of the invention anauto-focusing method for determining an in-focus position of a pluralityof wells in at least a portion of a multi-well plate, the methodincluding:

using a first objective lens having a first magnification to identify,in each of at least three wells of a selected subset of the plurality ofwells, an in-focus position of each said well with respect to the firstobjective lens;

on the basis of at least three said in-focus positions, computing aplane along which the at least three wells will be in focus with respectto at least one objective lens having a second magnification that is notgreater than the first magnification; and

using the at least one objective lens to scan, along the plane, at leastsome of the plurality of wells in the portion of the plate.

In some embodiments, the at least one objective lens is the firstobjective lens, and the first magnification is equal to the secondmagnification. In some embodiments, the computing a plane includescomputing a plane along which the at least three wells are in focus withrespect to the first objective lens.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, wherein thesecond magnification is smaller than the first magnification. In someembodiments, computing a plane includes translating at least three ofthe in-focus positions identified using the first objective lens tocorresponding second in-focus positions with respect to the secondobjective lens based on optical characteristics of the second objectivelens; and computing the plane on the basis of at least three the secondin-focus positions. In some embodiments, computing a plane includes: onthe basis of at least three said in-focus positions, computing a firstplane along which the at least three wells will be in focus with respectto the first objective lens; and translating the first plane to acorresponding plane along which the at least three wells will be infocus with respect to the second objective lens based on opticalcharacteristics of the second objective lens, thereby to compute theplane.

In some embodiments, scanning using the at least one objective lens iscarried out without carrying out additional focusing operations.

In some embodiments, the subset of the plurality of wells includes morethan three of the plurality of wells.

In some embodiments, identifying an in-focus position includesidentifying an in-focus position for each well in the subset.

In some embodiments, each of the wells includes (a) a generallycylindrical side wall, and (b) a bottom surface which includes a portionof at least one of a sphere, a paraboloid, and an ellipsoid. In someembodiments, each of the wells has a U-shaped cross-section.

In some embodiments, each of the wells includes a generally cylindricalside wall and a planar bottom surface. In some embodiments, the planarbottom surface lies generally parallel to a top surface of the multiwellplate, such that the well has a rectangular cross section.

In some embodiments, each of the wells is frusto-conical.

In some embodiments, each of the wells has an inclined side wall, aplanar bottom, and a trapezoidal cross section.

In some embodiments, the method further includes, prior to the using afirst objective lens, aligning the first objective lens to lie axiallyover the center of one of the wells.

In some embodiments, the portion of the plate includes a quadrant of theplate. In some embodiments, the portion of the plate includes anentirety of the plate.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furthercomprises, following the computing the plane and prior to the using theat least one objective lens:

storing alignment data of the plate along the X and Y axes in the firstscanning device, and data relating to the plane, in a computer storageelement which is in communication with the first and the second scanningdevices;

moving the multi-well plate to the second scanning device; and

aligning the multi-well plate along the X and Y axes in the secondscanning device based on the alignment data stored in the computerstorage element.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing method for determining an in-focus positionof at least a portion of a well in a plate, the method including: usinga first objective lens having a first magnification to identify, at atleast one location of the well, a first in-focus position of at least aportion of the well with respect to the first objective lens;identifying, for the first in-focus position, a corresponding in-focusposition with respect to at least one objective lens having a secondmagnification, based on optical characteristics of the at least oneobjective lens; and using the at least one objective lens to scan, at aheight corresponding to the corresponding in-focus position, at leastthe portion of the well, wherein the second magnification is not greaterthan the first magnification.

In some embodiments, the at least one objective lens is the firstobjective lens, the first magnification is equal to the secondmagnification, and the corresponding in-focus position is the firstin-focus position.

In some embodiments, the at least one objective lens includes a secondobjective lens, different from the first objective lens, wherein thesecond magnification is smaller than the first magnification. In somesuch embodiments, the identifying includes translating the firstin-focus position to the corresponding in-focus position with respect tothe second objective lens based on optical characteristics of the secondobjective lens.

In some embodiments, scanning using the at least one objective iscarried out without carrying out additional focusing operations.

In some embodiments, the well includes a generally cylindrical side walland a bottom surface which includes a portion of at least one of asphere, a paraboloid, and an ellipsoid. In some embodiments, the wellhas a U-shaped cross-section.

In some embodiments, the well includes a generally cylindrical side walland a planar bottom surface. In some embodiments, the planar bottomsurface lies generally parallel to a top surface of the plate, such thatthe well has a rectangular cross section.

In some embodiments, the well is frusto-conical. In some embodiments,the well has an inclined side wall, a planar bottom, and a trapezoidalcross section.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furthercomprises, following identifying the corresponding in-focus position andprior to the using the at least one objective lens:

-   -   storing alignment data relating to alignment of the plate along        the X and Y axes in the first scanning device, and data relating        to the corresponding in focus position, in a computer storage        element which is in communication with the first and the second        scanning devices;    -   moving the plate to the second scanning device; and    -   aligning the well in the plate in the second scanning device        based on the registration data stored in the computer storage        element.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the device including: a computationcomponent programmed to compute a plane along which at least three wellsin the portion of the plate would be in focus with respect to anobjective lens; a first objective lens functionally associated with thecomputation component, the first objective lens having a firstmagnification, signals obtained with the first objective lens being usedby the computation component for identifying an in-focus position foreach of at least three wells of a selected subset of the plurality ofwells; and at least one objective lens having a second magnification,the second magnification being not greater than the first magnification,for scanning at least some of the plurality of wells in the portion ofthe plate along the plane, wherein the computation component isconfigured to compute the plane along which the at least three wellswould be in-focus with respect to the at least one objective lens on thebasis of at least three the in-focus positions.

In some embodiments, the at least one objective lens is configured toscan the plurality of wells along the plane without carrying outadditional focusing operations.

In some embodiments, the at least one objective lens is the firstobjective lens, and the second magnification is equal to the firstmagnification.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, and the secondmagnification is smaller than the first magnification.

In some embodiments, the computation component is programmed to computethe in-focus plane by: translating at least three of the in-focuspositions identified using the first objective lens to correspondingsecond in-focus positions with respect to the second objective lensbased on optical characteristics of the second objective lens; andcomputing the in-focus plane on the basis of at least three the secondin-focus positions.

In some embodiments, the computation component is programmed to computethe in-focus plane by: on the basis of at least three the in-focuspositions, computing a first plane along which the at least three wellswill be in focus with respect to the first objective lens; andtranslating the first plane to a corresponding plane along which the atleast three wells will be in focus with respect to the second objectivelens based on optical characteristics of the second objective lens,thereby to compute the plane.

In some embodiments, the computation component is programmed to identifyan in-focus position for each well in the subset.

In some embodiments, the device is configured for use with a plate inwhich each of the wells includes (a) a generally cylindrical side wall,and (b) a bottom surface which includes at least one of a portion of asphere, a paraboloid, and an ellipsoid. In some embodiments, the deviceis configured for use with a plate in which each of the wells has aU-shaped cross section.

In some embodiments, the device is configured for use with a plate inwhich each of the wells includes a generally cylindrical side wall and aplanar bottom surface. In some embodiments, the planar bottom surfacelies generally parallel to a top surface of the plate, such that each ofthe wells has a generally rectangular cross section.

In some embodiments, the device is configured for use with a plate inwhich each of the wells is frusto-conical. In some embodiments, thedevice is configured for use with a plate in which each of the wells hasan inclined side wall, a planar bottom, and a trapezoidal cross section.

In some embodiments, the portion of the plate includes a quadrant of theplate. In some embodiments, the portion of the plate includes anentirety of the plate.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of at least a portion of a well, the device including:a computation component programmed to identify an in-focus position ofthe portion of the well; a first objective lens functionally associatedwith the computation component, the first objective lens having a firstmagnification, signals obtained with the first objective lens being usedby the computation component for identifying, in at least one positionof the well, a first in-focus position of the well with respect to thefirst objective lens; and at least one objective lens having a secondmagnification, the second magnification not being greater than the firstmagnification, for scanning at least a portion of the well at a heightof a corresponding in-focus position of the well with respect to the atleast one objective lens, wherein the computation component isprogrammed to identify the corresponding in-focus position based onoptical characteristics of the at least one objective lens.

In some embodiments, the at least one objective lens is configured toscan the portion of the well without carrying out additional focusingoperations.

In some embodiments, the at least one objective lens is the firstobjective lens, the second magnification is equal to the firstmagnification, and the corresponding in-focus position is the same asthe first in-focus position.

In some embodiments, the at least one objective lens is a secondobjective lens different from the first objective lens, and wherein thesecond magnification is smaller than the first magnification.

In some embodiments, the computation component is programmed to identifythe corresponding in-focus position by translating the first in-focusposition to the corresponding in-focus position with respect to thesecond objective lens based on optical characteristics of the secondobjective lens.

In some embodiments, the device is configured for use with a wellincluding a generally cylindrical side wall and a bottom surface whichincludes at least one of a portion of a sphere, a paraboloid, and anellipsoid. In some embodiments, the device is configured for use with awell having a U-shaped cross section.

In some embodiments, the device is configured for use with a wellincluding a generally cylindrical side wall and a planar bottom surface.In some embodiments, the planar bottom surface lies generally parallelto a top surface of the plate, such that the well has a generallyrectangular cross section.

In some embodiments, the device is configured for use with afrusto-conical well. In some embodiments, the device is configured foruse with a well having an inclined side wall, a planar bottom, and atrapezoidal cross section.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing system for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the system including:

a computation component programmed to compute a plane along which atleast three wells in the portion of the plate would be in focus withrespect to an objective lens;

a first scanning device including a first objective lens and beingfunctionally associated with the computation component, the firstobjective lens having a first magnification, signals obtained with thefirst objective lens being used by the computation component foridentifying an in-focus position for each of at least three wells of aselected subset of the plurality of wells;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining of the signals and datarelating to the computed plane; and

a second scanning device functionally associated with the storagecomponent and including at least one objective lens having a secondmagnification, the second magnification being not greater than the firstmagnification, for scanning at least some of the plurality of wells inthe portion of the plate along the plane,

wherein the second scanning device is configured to align the platealong X and Y axes according to the alignment data stored in the storagecomponent, and

wherein the computation component is configured to compute the planealong which the at least three wells would be in-focus with respect tothe at least one objective lens on the basis of at least three thein-focus positions.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of at least a portion of a well, the device including:

a computation component programmed to identify an in-focus position ofthe portion of the well;

a first scanning device including a first objective lens and beingfunctionally associated with the computation component, the firstobjective lens having a first magnification, signals obtained with thefirst objective lens being used by the computation component foridentifying, in at least one position of the well, a first in-focusposition of the well with respect to the first objective lens;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining of the signals and datarelating to the identified in-focus position of the portion of the well;and

a second scanning device functionally associated with the storagecomponent and including a second objective lens, the second objectivelens being different from the first objective lens and having a secondmagnification, the second magnification being smaller than the firstmagnification, for scanning at least a portion of the well at a heightof a corresponding in-focus position of the well with respect to the atleast one objective lens,

wherein the second scanning device is configured to align the platealong X and Y axes according to the alignment data stored in the storagecomponent, and

wherein the computation component is programmed to identify thecorresponding in-focus position based on optical characteristics of theat least one objective lens.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing method for determining an in-focus positionof a plurality of wells in at least a portion of a multi-well plate, themulti-well plate having a plate bottom surface and each well in theplurality of wells having a well bottom surface, the method including:

using a first objective lens having a first magnification to identify,for each well in a selected subset of the plurality of wells, a positionof the plate bottom surface with respect to the well;

using at least one objective lens having a second magnification that isnot greater than the first magnification, and beginning from theposition of the plate bottom surface with respect to each well in theselected subset, identifying an in-focus position of the well bottomsurface for each well with respect to the at least one objective lens;and

using the at least one objective lens to scan, at the in focus position,each of the wells in the selected subset.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furtherincludes, following using the first objective lens and prior to theusing the at least one objective lens:

storing alignment data relating to alignment of the plate along X and Yaxes in the first scanning device, and data relating to the identifiedposition of the plate bottom surface with respect to each well in theselected subset, in a computer storage element which is in communicationwith the first and the second scanning devices;

moving the multi-well plate to the second scanning device; and

aligning the multi-well plate along X and Y axes in the second scanningdevice based on the alignment data stored in the computer storageelement.

In some embodiments, the aligning further includes aligning the at leastone objective lens of the second scanning device at a height along the Zaxis of the second scanning device which is in focus with respect to theidentified position of the plate bottom surface.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing method for determining an in-focus positionof a plurality of wells in at least a portion of a multi-well plate, themulti-well plate having a plate bottom surface and each well in theplurality of wells having a well bottom surface, the method including:

using a first objective lens having a first magnification to identify,for each well in a selected subset of the plurality of wells, a firstposition of the plate bottom surface and a second position of a wellbottom surface with respect to the well;

based on the identified second position, identifying, for each well inthe selected subset, an in-focus position of the well relative to atleast one objective lens having a second magnification that is notgreater than the first magnification; and

using the at least one objective lens to scan, at the in focus positionof the well, each of the wells in the selected subset.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furtherincludes, following using the first objective lens and prior to theusing the at least one objective lens:

-   -   storing alignment data relating to alignment of the plate along        X and Y axes in the first scanning device, and data relating to        the identified position of the well bottom surface, in a        computer storage element which is in communication with the        first and the second scanning devices;    -   moving the multi-well plate to the second scanning device; and    -   aligning the multi-well plate along X and Y axes in the second        scanning device based on the alignment data stored in the        computer storage element.

In some embodiments, the aligning further includes aligning the at leastone objective lens of the second scanning device at a height along the Zaxis of the second scanning device which is in focus with respect to theidentified position of the well bottom surface.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, the method further including,following storing the alignment data, removing the multi-well plate fromthe scanning device, and moving the multi-well plate to the secondscanning device includes returning the multi-well plate to the scanningdevice.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, the method further including,following storing the alignment data, changing an objective lens of thescanning device from the first objective lens to the at least oneobjective lens.

In some embodiments, the first objective lens includes an air objectivelens, the at least one objective lens is a second objective lens whichis an oil immersion objective lens or a water immersion objective lens,and the identifying includes computing a difference between the secondposition and the in-focus position due to the first objective lens andthe second objective lens being of different types.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, and the secondmagnification is smaller than the first magnification, and theidentifying includes computing a difference between the second positionand the in-focus position due to a magnification difference between thefirst and second magnifications.

In some embodiments, the at least one objective lens is the firstobjective lens.

In some embodiments, the first magnification is equal to the secondmagnification.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, wherein thesecond magnification is smaller than the first magnification.

In some embodiments, the subset of the plurality of wells includes morethan three of the plurality of wells.

In some embodiments, each of the wells includes a generally cylindricalside wall, and a bottom surface including a portion of at least one of asphere, a paraboloid, and an ellipsoid. In some embodiments, each of thewells has a U-shaped cross-section.

In some embodiments, each of the wells includes a generally cylindricalside wall, and a planar bottom surface. In some embodiments, the planarbottom surface lies generally parallel to a top surface of the multiwellplate, such that the well has a rectangular cross section.

In some embodiments, each of the wells is frusto-conical. In someembodiments, each of the wells has an inclined side wall, a planarbottom, and a trapezoidal cross section.

In some embodiments, the method further includes, prior to using a firstobjective lens, aligning the first objective lens to lie axially overthe center of one of the wells.

In some embodiments, the portion of the plate includes a quadrant of theplate. In some embodiments, the portion of the plate includes anentirety of the plate.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing method for determining an in-focus positionof a plurality of fields in at least a portion of a well in a plate, theplate having a plate bottom surface and the well having a well bottomsurface, the method including:

using a first objective lens having a first magnification to identify,at each field in a selected subset of the plurality of fields, aposition of the plate bottom surface with respect to the field;

using a second objective lens having a second magnification that issmaller than the first magnification, and beginning from the position ofthe plate bottom surface with respect to each the field in the selectedsubset, identifying an in-focus position of the well bottom surface ofeach the field in the selected subset with respect to the secondobjective lens; and

using the second objective lens to scan, at the in-focus position of thewell bottom surface, each the field in the selected subset.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furtherincludes, following the using the first objective lens and prior to theusing the at least one objective lens:

storing alignment data relating to alignment of the plate along X and Yaxes in the first scanning device, and data relating to the identifiedposition of the plate bottom surface with respect to each the field inthe selected subset, in a computer storage element which is incommunication with the first and the second scanning devices;

moving the multi-well plate to the second scanning device; and

aligning the multi-well plate along X and Y axes in the second scanningdevice based on the alignment data stored in the computer storageelement.

In some embodiments, the aligning further includes aligning the at leastone objective lens of the second scanning device at a height along the Zaxis of the second scanning device which is in focus with respect to theidentified position of the plate bottom surface.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing method for determining an in-focus positionof a plurality of fields in at least a portion of a well in a plate, theplate having a plate bottom surface and the well having a well bottomsurface, the method including:

using a first objective lens having a first magnification to identify,at each field in a selected subset of the plurality of fields, a firstposition of the plate bottom surface and a second position of the wellbottom surface with respect to the field;

based on the identified second position, identifying, for each the fieldin the selected subset, an in-focus position of the field relative to atleast one objective lens having a second magnification that is notgreater than the first magnification; and

using the at least one objective lens to scan, at the in-focus positionof the field, each the field in the selected subset.

In some embodiments, using a first objective lens is carried out in afirst scanning device and using the at least one objective lens iscarried out in a second scanning device, and the method furtherincludes, following using the first objective lens and prior to theusing the at least one objective lens:

storing alignment data relating to alignment of the plate along X and Yaxes in the first scanning device, and data relating to the identifiedposition of the well bottom surface with respect to each the field inthe selected subset, in a computer storage element which is incommunication with the first and the second scanning devices;

moving the multi-well plate to the second scanning device; and

aligning the multi-well plate along X and Y axes in the second scanningdevice based on the alignment data stored in the computer storageelement.

In some embodiments, the aligning further includes aligning the at leastone objective lens of the second scanning device at a height along the Zaxis of the second scanning device which is in focus with respect to theidentified position of the well bottom surface.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, the method further includes,following storing the alignment data, removing the multi-well plate fromthe scanning device, and moving the multi-well plate to the secondscanning device includes returning the multi-well plate to the scanningdevice.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, the method further includes,following storing the alignment data, changing an objective lens of thescanning device from the first objective lens to the at least oneobjective lens.

In some embodiments, the first objective lens includes an air objectivelens, the at least one objective lens is a second objective lens whichis an oil immersion objective lens or a water immersion objective lens,and the identifying includes computing a difference in between thesecond position and the in-focus position due to the first objectivelens and the second objective lens being of different types.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, and the secondmagnification is smaller than the first magnification, and theidentifying includes computing a difference between the second positionand the in-focus position due to a magnification difference between thefirst and second magnifications.

In some embodiments, the well includes a generally cylindrical sidewall, and a bottom surface including a portion of at least one of asphere, a paraboloid, and an ellipsoid. In some embodiments, the wellhas a U-shaped cross-section.

In some embodiments, the well includes a generally cylindrical sidewall, and a planar bottom surface. In some embodiments, the planarbottom surface lies generally parallel to a top surface of the plate,such that the well has a rectangular cross section.

In some embodiments, the well is frusto-conical. In some embodiments,the well has an inclined side wall, a planar bottom, and a trapezoidalcross section.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the plate having a plate bottom surface andeach of the plurality of wells having a well bottom surface, the deviceincluding:

a computation component programmed to identify a position of the platebottom surface with respect to each well in a selected subset of theplurality of wells and a position of the well bottom surface withrespect to each well in the selected subset;

a first objective lens functionally associated with the computationcomponent, the first objective lens having a first magnification,signals obtained with the first objective lens being used by thecomputation component for identifying the position of the plate bottomsurface with respect to each well in the subset; and

at least one objective lens having a second magnification, the secondmagnification being not greater than the first magnification, signalsobtained with the at least one objective lens being used by thecomputation component for identifying the position of the well bottomsurface with respect to each well in the subset, the at least oneobjective lens configured for scanning each well in the subset at theposition of the well bottom surface.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the plate having a plate bottom surface andeach of the plurality of wells having a well bottom surface, the deviceincluding:

a computation component programmed to identify a position of the platebottom surface with respect to each well in a selected subset of theplurality of wells and a position of the well bottom surface withrespect to each well in the selected subset;

a first objective lens functionally associated with the computationcomponent, the first objective lens having a first magnification,signals obtained with the first objective lens being used by thecomputation component for identifying the positions of the plate bottomsurface and of the well bottom surface with respect to each well in thesubset; and

at least one objective lens having a second magnification, the secondmagnification being not greater than the first magnification, the atleast one objective lens configured for scanning each well in the subsetat the position of the well bottom surface.

In some embodiments, the at least one objective lens is the firstobjective lens and the second magnification is equal to the firstmagnification.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, and the secondmagnification is smaller than the first magnification.

In some embodiments, the second objective lens is an oil immersionobjective lens, and the computation component is programmed to compute,based on at least one of the positions of the plate bottom surface andthe well bottom surface identified with respect to the first objectivelens, at least one of a corresponding position of the plate bottomsurface and the well bottom surface with respect to the oil immersionobjective lens.

In some embodiments, the second objective lens is an water immersionobjective lens, and the computation component is programmed to compute,based on at least one of the positions of the plate bottom surface andthe well bottom surface identified with respect to the first objectivelens, at least one of a corresponding position of the plate bottomsurface and the well bottom surface with respect to the water immersionobjective lens.

In some embodiments, the device is configured for use with a plate inwhich each of the wells includes a generally cylindrical side wall, anda bottom surface including at least one of a portion of a sphere, aparaboloid, and an ellipsoid. In some embodiments, the device isconfigured for use with a plate in which each of the wells has aU-shaped cross section.

In some embodiments, the device is configured for use with a plate inwhich each of the wells includes a generally cylindrical side wall, anda planar bottom surface. In some embodiments, the planar bottom surfacelies generally parallel to a top surface of the plate, such that each ofthe wells has a generally rectangular cross section.

In some embodiments, the device is configured for use with a plate inwhich each of the wells is frusto-conical. In some embodiments, thedevice is configured for use with a plate in which each of the wells hasan inclined side wall, a planar bottom, and a trapezoidal cross section.

In some embodiments, the portion of the plate includes a quadrant of theplate.

In some embodiments, the portion of the plate includes an entirety ofthe plate.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of a plurality of fields in at least a portion of awell in a plate, the plate having a plate bottom surface and the wellhaving a well bottom surface, the device including:

a computation component programmed to identify, for each field in aselected subset of the plurality of fields, a first position of theplate bottom surface and a second position of the well bottom surfacewith respect to the field;

a first objective lens functionally associated with the computationcomponent, the first objective lens having a first magnification,signals obtained with the first objective lens being used by thecomputation component for identifying, in at least one field of selectedsubset of fields, a position of the plate bottom surface; and

at least one objective lens having a second magnification, the secondmagnification being not greater than the first magnification, signalsobtained with the at least one objective lens being used by thecomputation component for identifying the position of the well bottomsurface with respect to each the field in the subset, the at least oneobjective lens configured for scanning each the field in the subset atthe position of the well bottom surface with respect to the field.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing device for automatically determining anin-focus position of a plurality of fields in at least a portion of awell in a plate, the plate having a plate bottom surface and the wellhaving a well bottom surface, the device including:

a computation component programmed to identify, for each field in aselected subset of the plurality of fields, a first position of theplate bottom surface and a second position of the well bottom surfacewith respect to the field;

a first objective lens functionally associated with the computationcomponent, the first objective lens having a first magnification,signals obtained with the first objective lens being used by thecomputation component for identifying the positions of the plate bottomsurface and of the well bottom surface with respect to each the field inthe subset; and

at least one objective lens having a second magnification, the secondmagnification being not greater than the first magnification, the atleast one objective lens configured for scanning each the field in thesubset at the position of the well bottom surface.

In some embodiments, the at least one objective lens is the firstobjective lens and the second magnification is equal to the firstmagnification.

In some embodiments, the at least one objective lens is a secondobjective lens, different from the first objective lens, and the secondmagnification is smaller than the first magnification.

In some embodiments, the second objective lens is an oil immersionobjective lens, and the computation component is programmed to compute,based on at least one of the positions of the plate bottom surface andthe well bottom surface identified with respect to the first objectivelens, at least one of a corresponding position of the plate bottomsurface and the well bottom surface with respect to the oil immersionobjective lens.

In some embodiments, the second objective lens is a water immersionobjective lens, and the computation component is programmed to compute,based on at least one of the positions of the plate bottom surface andthe well bottom surface identified with respect to the first objectivelens, at least one of a corresponding position of the plate bottomsurface and the well bottom surface with respect to the water immersionobjective lens.

In some embodiments, the device is configured for use with a wellincluding a generally cylindrical side wall, and a bottom surfaceincluding at least one of a portion of a sphere, a paraboloid, and anellipsoid. In some embodiments, the device is configured for use with awell having a U-shaped cross section.

In some embodiments, the device is configured for use with a wellincluding a generally cylindrical side wall, and a planar bottomsurface. In some embodiments, the planar bottom surface lies generallyparallel to a top surface of the plate, such that the well has agenerally rectangular cross section.

In some embodiments, the device is configured for use with afrusto-conical well. In some embodiments, the device is configured foruse with a well having an inclined side wall, a planar bottom, and atrapezoidal cross section.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing system for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the plate having a plate bottom surface andeach of the plurality of wells having a well bottom surface, the systemincluding:

a computation component programmed to identify a position of the platebottom surface with respect to each well in a selected subset of theplurality of wells and a position of the well bottom surface withrespect to each well in the selected subset;

a first scanning device functionally associated with the computationcomponent, and including a first objective lens having a firstmagnification, signals obtained with the first objective lens being usedby the computation component for identifying the position of the platebottom surface with respect to each well in the subset;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining if the signals and datarelating to the identified position of the plate bottom surface for eachwell in the subset; and

a second scanning device functionally associated with the storagecomponent and including at least one objective lens having a secondmagnification, the second magnification being not greater than the firstmagnification, signals obtained with the at least one objective lensbeing used by the computation component for identifying the position ofthe well bottom surface with respect to each well in the subset, the atleast one objective lens configured for scanning each well in the subsetat the position of the well bottom surface,

wherein the second scanning device is configured to align the platealong X and Y axes in the second scanning device based on the alignmentdata stored in the storage component.

In some embodiments, the second scanning device is further configured toalign the at least one objective lens at a height along the Z axis ofthe second scanning device which is in focus with respect to theidentified position of the plate bottom surface.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing system for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the plate having a plate bottom surface andeach of the plurality of wells having a well bottom surface, the systemincluding:

a computation component programmed to identify a position of the platebottom surface with respect to each well in a selected subset of theplurality of wells and a position of the well bottom surface withrespect to each well in the selected subset;

a first scanning device functionally associated with the computationcomponent, and including a first objective lens having a firstmagnification, signals obtained with the first objective lens being usedby the computation component for identifying the positions of the platebottom surface and of the well bottom surface with respect to each wellin the subset;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining of the signals and datarelating to the identified position of the plate bottom surface and ofthe well bottom surface for each well in the subset; and

a second scanning device functionally associated with the storagecomponent and including at least one objective lens having a secondmagnification, the second magnification being not greater than the firstmagnification, the at least one objective lens configured for scanningeach well in the subset at the position of the well bottom surface,

wherein the second scanning device is configured to align the platealong X and Y axes in the second scanning device based on the alignmentdata stored in the storage component.

In some embodiments, the second scanning device is further configured toalign the at least one objective lens at a height along the Z axis ofthe second scanning device which is in focus with respect to theidentified position of the well bottom surface.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, and the scanning device isconfigured to align the plate along the X and Y axes following removalof the plate from the scanning device and reinsertion of the plate intothe scanning device.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing system for automatically determining anin-focus position of a plurality of fields in at least a portion of awell in a plate, the plate having a plate bottom surface and each of theplurality of wells having a well bottom surface, the system including:

a computation component programmed to identify, for each field in aselected subset of the plurality of fields, a first position of theplate bottom surface with respect to the field and a second position ofthe well bottom surface with respect to the field;

a first scanning device functionally associated with the computationcomponent, and including a first objective lens having a firstmagnification, signals obtained with the first objective lens being usedby the computation component for identifying the position of the platebottom surface with respect to each the field in the subset;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining of the signals and datarelating to the identified position of the plate bottom surface for eachfield in the subset; and

a second scanning device functionally associated with the storagecomponent and including at least one objective lens having a secondmagnification, the second magnification being not greater than the firstmagnification, signals obtained with the at least one objective lensbeing used by the computation component for identifying the position ofthe well bottom surface with respect to each the field in the subset,the at least one objective lens configured for scanning each the fieldin the subset at the position of the well bottom surface,

wherein the second scanning device is configured to align the platealong X and Y axes in the second scanning device based on the alignmentdata stored in the storage component.

In some embodiments, the second scanning device is further configured toalign the at least one objective lens at a height along the Z axis ofthe second scanning device which is in focus with respect to theidentified position of the plate bottom surface.

There is also provided, in accordance with an embodiment of theinvention, an auto-focusing system for automatically determining anin-focus position of a plurality of fields in at least a portion of awell in a plate, the plate having a plate bottom surface and each of theplurality of wells having a well bottom surface, the system including:

a computation component programmed to identify, for each field in aselected subset of the plurality of fields, a first position of theplate bottom surface with respect to the field and a second position ofthe well bottom surface with respect to the field;

a first scanning device functionally associated with the computationcomponent, and including a first objective lens having a firstmagnification, signals obtained from the first objective lens being usedby the computation component for identifying the positions of the platebottom surface and of the well bottom surface with respect to each thefield in the subset;

a storage component functionally associated with the computationcomponent and with the first scanning device, configured to storealignment data relating to alignment of the plate along X and Y axes inthe first scanning device during obtaining of the signals and datarelating to the identified position of the plate bottom surface and ofthe well bottom surface for each the field in the subset; and

a second scanning device functionally associated with the storagecomponent and including at least one objective lens having a secondmagnification, the second magnification being not greater than the firstmagnification, the at least one objective lens configured for scanningeach the field in the subset at the position of the well bottom surface,

wherein the second scanning device is configured to align the platealong X and Y axes in the second scanning device based on the alignmentdata stored in the storage component.

In some embodiments, the second scanning device is further configured toalign the at least one objective lens at a height along the Z axis ofthe second scanning device which is in focus with respect to theidentified position of the well bottom surface.

In some embodiments, the first scanning device and the second scanningdevice are the same scanning device, and the scanning device isconfigured to align the plate along the X and Y axes following removalof the plate from the scanning device and reinsertion of the plate intothe scanning device.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In case of conflict, thespecification, including definitions, will take precedence.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. These terms encompass the terms “consisting of” and“consisting essentially of”.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

Embodiments of methods and/or devices of the invention may involveperforming or completing selected tasks manually, automatically, or acombination thereof. Some embodiments of the invention are implementedwith the use of components that comprise hardware, software, firmware orcombinations thereof. In some embodiments, some components aregeneral-purpose components such as general purpose computers ormonitors. In some embodiments, some components are dedicated or customcomponents such as circuits, integrated circuits or software.

For example, in some embodiments, some of an embodiment is implementedas a plurality of software instructions executed by a data processor,for example which is part of a general-purpose or custom computer. Insome embodiments, the data processor or computer comprises volatilememory for storing instructions and/or data and/or a non-volatilestorage, for example, a magnetic hard-disk and/or removable media, forstoring instructions and/or data. In some embodiments, implementationincludes a network connection. In some embodiments, implementationincludes a user interface, generally comprising one or more of inputdevices (e.g., allowing input of commands and/or parameters) and outputdevices (e.g., allowing reporting parameters of operation and results.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are described herein with reference tothe accompanying figures. The description, together with the figures,makes apparent to a person having ordinary skill in the art how someembodiments of the invention may be practiced. The figures are for thepurpose of illustrative discussion and no attempt is made to showstructural details of an embodiment in more detail than is necessary fora fundamental understanding of the invention. For the sake of clarity,some objects depicted in the figures are not to scale.

In the Figures:

FIGS. 1A and 1B are, respectively, a top plan view of a multiwell plateand a sectional view of a single well in a multiwell plate, the wellhaving a non-planar bottom surface, for which embodiments of theteachings herein may be useful;

FIG. 2 is a block diagram of an embodiment of a scanning device forauto-focusing on samples in a multiwell plate in accordance with anembodiment of the teachings herein;

FIG. 3 is a flow chart of an embodiment of a method for auto-focusing ascanning device on samples in a multiwell plate in accordance with anembodiment of the teachings herein;

FIG. 4 is a block diagram of an embodiment of a scanning system forauto-focusing on samples in a multiwell plate in accordance with anotherembodiment of the teachings herein; and

FIGS. 5A and 5B are flow charts of two embodiments of a method forauto-focusing a scanning device of a scanning system on samples in amultiwell plate in accordance with embodiments of the teachings herein.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The principles, uses and implementations of the teachings herein may bebetter understood with reference to the accompanying description andfigures. Upon perusal of the description and figures present herein, oneskilled in the art is able to implement the invention without undueeffort or experimentation.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in itsapplications to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples. The invention can beimplemented with other embodiments and can be practiced or carried outin various ways. It is also understood that the phraseology andterminology employed herein is for descriptive purpose and should not beregarded as limiting.

Reference is now made to FIGS. 1A and 1B which are, respectively, a topplan view of a multiwell plate and a sectional view of a single well ina multiwell plate, the well having a non-planar bottom surface, forwhich embodiments of the teachings herein may be useful.

As seen in FIG. 1A, a multiwell plate 10 has a top surface 11, sidesurfaces (not shown) and, in some embodiments, a bottom surface (notshown). The plate 10 includes a plurality of wells 12, arranged in agrid formed of columns 14 and rows 16 and accessible via apertures 17 intop surface 11. Typically, the rows and columns are enumerated orotherwise labeled so as to enable a user to easily reference a specificwell 12. The multiwell plate 10 in the illustrated embodiment includes96 wells, though other types of plates, which include, for example, adifferent number of wells, such as 12, 24, or 384 wells, may be usedwith the teachings herein as described in further detail hereinbelow.Typically, the wells 12 have fixed distances between one another, andthus are distributed on plate 10 at regular intervals. Specifications asto the distanced between wells are standard in the art, and aretypically also provided by the manufacturer of the plate. Often, thenumber of wells in the plate have a 3:2 aspect ratio. As such the wellsmay be arranged, for example, as a 3×2 grid, 6×4 grid, 12×8 grid, or24×16 grid.

Turning to FIG. 1B, it is seen that the cross section of each well 12 inthe plate 10 may be non-rectangular, such that the well has a non-linearbottom surface. In the illustrated embodiment, the well 12 includes acavity 18 and has a U-shaped cross section, such that side walls 20 ofthe well generally form a cylinder, and a bottom portion 22 of the wellforms part of a sphere, part of a paraboloid, or part of an ellipsoid,thereby defining a curved bottom surface to the well. As such, the welltypically has a U-shaped cross-section or a cross-section somewhatresembling a parabola. Typically, the thickness of side walls 20 and ofbottom portion 22 is uniform. A rim 26, typically forming part of, andbeing flush with or being raised with respect to, top surface 11 ofplate 10, often surrounds the well 12.

Multiwell plates including wells having non-planar bottom surfaces arewell known in the art, and are commercially available from manymanufacturers, such as Corning Incorporated Life Sciences of Tewksbury,Mass. Such multi-well plates are used for many types of samples,including for growing spheroids, for growing non-adherent cells such aslymphocytes and other blood cells, for analysis of 3-dimensional samplesand for handling of compounds. Oftentimes, analysis of such samplesrequires imaging of the samples within the wells.

It will be appreciated that due to the curvature of the bottom surfaceof well 12 the area at which a microscope viewing the well would be infocus is typically very small, and in some cases comprises a singlepoint. As such, existing auto-focusing mechanisms, such as thatdisclosed in U.S. Pat. No. 7,109,459 often do not succeed in focusing ona sample disposed within the well. As explained hereinbelow, the methodof the teachings herein enables an operator to autofocus a scanningdevice on a well having a non-planar bottom, such as U-shaped wells 12of FIG. 1B, without having to manually focus the scanning device on eachindividual well.

It will be appreciated that though the exemplary illustration showswells having a U-shaped cross section, the method of the teachingsherein as described hereinbelow may be used for other types of wells,such as wells having a planar bottom surface and a rectangular crosssection, or wells of a frusto-conical shape, i.e. comprising a cutoffcone having inclined side walls and a planar bottom, and having agenerally trapezoidal cross section.

Reference is now made to FIG. 2, which is a block diagram of anembodiment of a scanning device 200 for auto-focusing on wells in amultiwell plate in accordance with an embodiment of the teachingsherein.

It will be appreciated that the disclosure herein discussesauto-focusing on wells including samples as an example only, and thatthe same method and device may also be used to auto-focus on wells notcontaining a sample, or on a multiwell plate in which some wells includea sample and other wells do not.

As seen in FIG. 2, scanning device 100 includes a scanning microscope202, functionally associated with a sample platform movable along the X,Y, and Z axes. The sample platform is configured to have disposedthereon a sample plate 205, which may be for example a plate like plate10 of FIGS. 1A and 1B.

Microscope 202 further includes a plurality of objective lenses 206functionally associated with an objective lens exchanger 208. At anygiven time, a single one of lenses 206 is aligned with a sample platform(not shown) and is operational, such that the sample plate disposed onthe sample platform may be viewed through the objective lens. Objectivelens exchanger 208 is configured to change the operational lens, usedfor viewing the sample, when a change of objective is required. Anexample of such an exchanger is described, for example, in WO2012/097191, the contents of which are incorporated herein by reference.

Microscope 202 is functionally associated with at least one illuminationsource, controlled by a controlling unit (not shown). In someembodiments, the microscope includes a first illumination sourcecomprising a transmission light source 210 a, such as an LED lamp,configured to illuminate the sample platform during capturing signalsfrom a sample plate 205 disposed thereon.

In some embodiments, the microscope further includes a secondillumination source comprising an excitation light source 210 bconfigured to provide illumination to yield a response in a samplecarried on or in the sample plate 205, such as providing illumination toexcite a fluorescent or a luminescent component of the sample. In someembodiments, illumination from light source(s) 210 a and/or 210 bimpinges upon one or more optical elements 212, such as a mirror, adichroic cube, a beam splitter, a filter, and the like, prior toimpinging upon a sample disposed on the sample plate 205. In someembodiments, illumination from illumination source(s) 210 travelsthrough an optic fiber 213 before impinging on the sample.

In the context of the present application, the term “signals” relates toany illumination signals which may be obtained from the sample plate,including, but not limited to, laser reflection signals, laserrefraction signals, and images obtained by an imaging mechanism of ascanning device as described.

In some embodiments, the image visible by microscope 202 is captured byan image capturing unit (not shown), and is transferred to a processingunit 214 for further processing and analysis.

Reference is now made to FIG. 3, which is a flow chart of an embodimentof a method for auto-focusing a scanning device on samples in amultiwell plate in accordance with an embodiment of the teachingsherein.

The method described hereinbelow may be used in a scanning device, suchas scanning device 200 of FIG. 2, to automatically determine an in-focusposition of a plurality of samples disposed in a sample plate, such asplate 10 of FIG. 1A, the plate containing a plurality of wells. Themethod may be carried out on a plate including wells having a non-planarbottom surface, such as wells 12 of FIG. 1B, or on other types of wells,such as wells having a planar bottom surface, or frusto-conical wellshaving inclined side walls and a planar bottom, and the like.

As seen at step 300, a subset of the wells in the plate is selected. Insome embodiments, the subset includes at least three wells that eachcontains a liquid or a sample, though this is not necessary for themethod disclosed herein. For at least three of the wells in the subset,and in some embodiments for all the wells in the subset, an in-focusposition of the sample included in the well is identified with respectto a first objective lens having a first magnification, such as anobjective lens 206 of FIG. 2, at step 302.

Typically, the first objective lens has a fairly large magnification,such as for example 20×, 10× or the like.

In some embodiments, the subset includes more than three wells, butin-focus positions are identified only for three of the wells in thesubset. In some embodiments, the subset includes more than three wells,and in-focus positions are identified for more than three wells in thesubset, but not for all the wells in the subset. For example, the subsetmay contain at least five wells, and in-focus positions are identifiedfor at least four wells but not for all the wells in the subset. In someembodiments, in-focus positions are identified for all the wells in thesubset.

The in-focus positions of the samples in the wells of the subset may beidentified using any suitable method known in the art, including bothmanual and automatic methods. In some embodiments, the in-focuspositions are identified substantially as described in U.S. Pat. No.7,109,459, which is incorporated by reference as if fully set forthherein.

In accordance with the teachings of U.S. Pat. No. 7,109,459, in order toidentify the in-focus positions, the focal plane of the first objectiveis spaced from a surface of the plate, such as a bottom surface of theplate, a certain distance, for example about one millimeter. The focalplane of the objective is then displaced towards the plate, for exampleby displacing the objective or the plate relative to one another. Forexample, the objective lens may be disposed below the plate, such thatthe focal plane of the objective lens is disposed below the surface ofthe plate and the focal plane is displaced vertically upward toward thesurface of the plate.

During displacement of the focal plane of the objective lens, controlhardware of the microscope records the intensity of light reflected fromthe plate, until the intensity of the detected light reaches a maximalvalue, which, in some embodiments, is higher than a preset threshold.This maximal value of the detected light intensity corresponds to anin-focus position of a surface of the plate.

Without wishing to be bound by theory, in the example described above,in which the objective lens is disposed below the plate and the focalplane is initially disposed below the plate and is displaced toward theplate, it is believed that the location at which maximal light intensityis observed corresponds to a point at which the focal plane of theobjective lens is tangential to the curved surface of the well bottom.

Subsequently, in some embodiments, the focal plane of the objectivecontinues to be displaced toward the plate, until another peak in theintensity of reflected light is detected, the peak being defined by arespective threshold value in accordance with the environment and thesample being tested. Without wishing to be bound by theory, in theexample described above, in which the objective lens is disposed belowthe plate and the focal plane is initially disposed below the plate andis displaced toward the plate, it is surmised that this second peak inthe intensity of the reflected light occurs when the focal plane of theobjective lens is tangent to the intra-well plate bottom, and typicallyrepresents an offset from an in-focus position of the sample. Themagnitude of the offset may be determined manually by the user, or maybe determined automatically using methods known in the art.

In some embodiments, the offset is computed from the first peak in theintensity of detected light, without continuing the search for a secondpeak in the intensity of detected light. In such embodiments, themagnitude of the offset may be determined manually by the user, or maybe determined automatically using methods known in the art.

It will be appreciated that the direction in which the focal plane isdisplaced toward the plate, and the order in which the peaks inintensity of detected light are identified, is dependent on the setup ofthe scanning device. For example, in some embodiments, the objectivelens is disposed below the sample plate, but the focal plane of theobjective lens is disposed above the well bottom, such that the focalplane would be displaced downward toward the well bottom.

Without wishing to be bound by theory, in such embodiments, it issurmised that the first peak in the intensity of the reflected lightoccurs when the focal plane of the objective lens is tangent to theintra-well plate bottom, and typically represents an offset from anin-focus position of the sample while the location at which the secondpeak in intensity of the reflected light is detected corresponds to apoint at which the focal plane of the objective lens is tangential tothe curved surface of the well bottom. A corresponding situation occursin other embodiments in which the objective lens is disposed above thesample plate, and the focal plane of the objective lens is disposedabove the well bottom, such that the focal plane would be displaceddownward toward the well bottom.

As another example, in some embodiments, the objective lens is disposedabove the sample plate, but the focal plane of the objective lens isdisposed below the well bottom, such that the focal plane would bedisplaced upward toward the well bottom. Without wishing to be bound bytheory, in such embodiments, it is surmised that the location at whichthe first peak in intensity of the reflected light is detectedcorresponds to a point at which the focal plane of the objective lens istangential to the curved surface of the well bottom while the secondpeak in the intensity of the reflected light occurs when the focal planeof the objective lens is tangent to the intra-well plate bottom, andtypically represents an offset from an in-focus position of the sample.

In some embodiments, the center of the well, at which the in-focusposition would lie, is identified based on the plate specificationsprovided by the manufacturer. In some embodiments, the center of thewell is determined using X-Y displacement of the plate or X-Ydisplacement of the objective lens, until the center of a well or theedge of a well are identified using suitable light detection parametersand characteristics, as is known in the art.

At step 304, at least three of the in-focus positions identified at step302 are used to compute a plane along which at least some of theplurality of wells in the plate, and typically all the wells in theplate, are in-focus or close to in-focus with respect to a secondobjective lens, such as an objective lens 206 of FIG. 2. The secondobjective lens has a second magnification which is not greater than thefirst magnification of the first objective lens. As describedhereinbelow, the wells are scanned using the second objective lens basedon the location computed plane, by maintaining the position of thesecond objective lens during scanning so that for any given wellscanned, the calculated plane and the focal plane of the secondobjective lens are coincident or close to coincident.

In some embodiments, the plane is computed by translating at least threeof, and typically each of, the in-focus positions identified in step 302using the first objective lens to corresponding second in-focuspositions with respect to the second objective lens, based on opticalcharacteristics of the second objective lens, and computing a planeincluding at least three of the second in-focus positions.

In some embodiments, the plane is computed by computing, on the basis ofat least three of the in-focus positions, a first plane along which atleast some of the plurality of wells in the plate, and typically all thewells in the plate, are in-focus or close to in-focus with respect tothe first objective lens. The first plane is then translated into thecorresponding plane along which at least some of the wells, andtypically all the wells, are in-focus or close to in-focus with respectto the second objective lens, based on optical characteristics of thesecond objective lens.

As mentioned above, the second objective lens has a magnification thatis not greater than the first magnification of the first objective lens.As such, in some embodiments the second magnification is smaller thanthe first magnification, and may be, for example, 4× or 2×. In someembodiments, the second magnification is equal to the firstmagnification, but the numerical aperture value of the second objectivelens is higher than the numerical aperture value of the first objectivelens.

In some embodiments, the plane is computed using all the in-focuspositions identified at step 302. In other embodiments, the plane iscomputed using fewer than all the in-focus positions identified at step302.

In some embodiments, the plane is computed for a section of the plate,for example for a quadrant, using at least three in-focus positionsidentified, using the first objective lens, within that section of theplate. In such embodiments, the method described herein is repeated foreach section or quadrant of the plate using a different set of in-focuspositions for each such section.

At step 306, which may occur before or after step 304 above, the firstobjective lens is changed to the second objective lens, for example by asuitable hardware mechanism such as objective lens exchanger 208 of FIG.2. In some embodiments the first and second objective lenses areidentical, and step 306 of FIG. 3 is omitted.

Finally, at step 308, the wells of the multiwell plate are scanned, orimaged, along the plane computed at step 304, using the second objectivelens, without carrying out any additional focusing operations.

The scanning at step 308 may be carried out using any suitable methodknown in the art, including capturing an image stack, which isparticularly useful when imaging a three dimensional construct such as aspheroid.

In some embodiments, the teachings herein may be carried out on a platehaving a single well, or on a single well within a multi-well plate. Insuch embodiments, the first objective is used to find an in-focus pointof the sample in the plate. The in-focus point found using the firstobjective is translated into an in-focus point for the second objective,based on the optical characteristics of the second objective. The secondobjective is then used to scan the plate, when placed at the translatedin-focus point, or at the height thereof.

As mentioned hereinabove, in some embodiments, it is advantageous toinitially perform pre-scanning, or mapping, of the topography of amulti-well or other plate prior to conducting the scanning describedherein. For example, when using a sophisticated scanning machine, suchpre-scanning may be carried out in a simpler machine and reduce the timethe scanning machine spends on focusing operations, thereby increasingits productivity.

As another example, in some cases, it is desirable to carry out theactual scanning using an oil immersion objective lens or a waterimmersion objective lens, so as to be able to use a higher numericalaperture during the scanning and to reduce refraction between the lensand the liquid medium of a sample. As is well known in the art, an oilimmersion objective lens is an objective lens, which has been designedfor use with oil, rather than air, as the medium between the lens of theobjective and the surface of the imaged sample. Similarly, a waterimmersion objective lens is an objective lens which has been designedfor use with water, rather than air, as the medium between the lens ofthe objective and the surface of the imaged sample. Consequently, incontrast to a standard objective lens which is used with air between thelens of the objective and the imaged sample, an oil immersion objectivelens or a water immersion objective lens will have high power and ashort focal length, and will not be suitable for use as an objective inthe absence of oil or water having the refractive index with which theobjective was designed to work.

As mentioned above, oil immersion lenses and water immersion lenses areless suitable for prior art autofocusing methodologies. This is due tothe fact that the similar refraction index between the lens and theliquid medium of the sample causes there to be no focusing peak at thewell bottom surface (the transition between the plastic of the plate andthe liquid of the sample). In such cases, it is helpful to carry outinitial auto-focusing onto the plate bottom surface, using an airobjective lens. The plate may then be transferred to a device includingthe oil immersion lens, and focusing the oil immersion lens in astep-by-step fashion from the position of the plate bottom surfaceidentified in the initial mapping.

Reference is now made to FIG. 4, which is a block diagram of anembodiment of an scanning system 400 for auto-focusing on wells in amultiwell plate in accordance with an embodiment of the teachingsherein.

As seen in FIG. 4, the system includes a first scanning device 402,similar to device 200 described hereinabove with respect to FIG. 2. Asdescribed hereinabove, the first scanning device 402 may include ascanning microscope and may be functionally associated with a sampleplatform movable along the X, Y, and Z axes. The sample platform isconfigured to have disposed thereon a sample plate 405, held in apredetermined three dimensional position by at least one dedicated plateholder 406.

As described hereinabove, the microscope includes at least one objectivelens, and in some cases a plurality of objective lenses functionallyassociated with an objective lens exchanger, such as that shown in U.S.Pat. No. 9,170,412, the contents of which are incorporated herein byreference. The microscope is functionally associated with at least oneillumination source, controlled by a controlling unit (not shown). Insome embodiments, the microscope includes a first (single) illuminationsource configured to illuminate the sample on the sample platform duringimaging of the sample plate; in some embodiments the microscope includesa first illumination source configured to illuminate the sample on thesample platform during focusing and imaging of the sample plate andfurther includes a second illumination source comprising an excitationlight source configured to provide illumination to yield a response in asample carried on or in the sample plate, such as providing illuminationto excite a fluorescent or a luminescent component of the sample.

In some embodiments, illumination from the light source(s) impinges uponone or more optical elements, such as a mirror, a dichroic cube, a beamsplitter, a filter, and the like, prior to impinging upon a sampledisposed on the sample plate. In some embodiments, illumination fromillumination source(s) travels through an optic fiber before impingingon the sample, all as described hereinabove.

In some embodiments, the illumination signals captured by the microscopeof device 402 are transferred to a processing unit 414 for furtherprocessing and analysis. Processing unit 414 is configured to identify,based on the signals obtained from first scanning device 402, a positionof a plate bottom surface and/or a well bottom surface in at least onewell or field of a well of the sample plate 405. In some embodiments,the illumination signals may be laser reflection signals or laserrefraction signals, whereas in other embodiments, in which the device402 includes an image capturing unit (not shown), the illuminationsignals may be images captured by the image capturing unit.

In some embodiments, the system 400 further includes a storage unit,such as a computer storage element 416, in communication with the firstscanning device 402 and with processing unit 414. The storage element416 is configured to store the position of the plate bottom surfaceand/or of the well bottom surface along the Z axis, or a distancebetween the objective lens and the plate bottom surface/well bottomsurface, as identified by processing unit 414, as well as thecoordinates of the sample plate within first scanning device 402 alongthe X and Y axes.

In some embodiments, system 400 further includes a second scanningdevice 420, functionally associated with storage unit 416 and withprocessing unit 414, the second scanning device 420 being substantiallysimilar to device 200 described hereinabove with respect to FIG. 2, andincluding similar components.

Specifically, second scanning device 420 includes at least one plateholder 426 similar to the plate holder(s) of first scanning device 402.When the plate is held by plate holder(s) 426 in second scanning device420, the information stored in the storage element 416 may be used toalign the plate along the X and Y axes so that the plate in the secondscanning device 420 is in the same position as it was duringpre-scanning in first scanning device 402. Additionally, the informationstored in storage element 416 may be used to align the objective lens ofscanning device 426 at a position along the Z axis which corresponds to,or is based on, the Z position of the plate bottom surface or wellbottom surface as determined by the first scanning device.

Signals, including images captured by second scanning device 420 aretransmitted to processing unit 414 for processing thereof.

In some embodiments, first and second scanning devices 402 and 420 sharea single processing unit 414, as illustrated in FIG. 4. In some suchembodiments, the single processing unit 414 may be a component of athird device of the system 400, separate from the first and secondscanning devices, as shown in FIG. 4. In other embodiments, the singleprocessing unit may form part of the first scanning device 402 and be incommunication with second scanning device 420, or may form part of thesecond scanning device 420 and be in communication with first scanningdevice 402.

In other embodiments, each of the first and second scanning devices maybe associated with a dedicated processing unit forming part of thescanning device.

Reference is now made to FIGS. 5A and 5B, which are flow charts of twoembodiments of a method for auto-focusing a scanning device of ascanning system on samples in a multiwell plate in accordance withembodiments of the teachings herein.

The methods described hereinbelow may be carried out in a scanningsystem, such as scanning system 400 of FIG. 4, to automaticallydetermine an in-focus position of a plurality of samples disposed in asample plate containing a plurality of wells.

Referring specifically to FIG. 5A, as seen at step 500, a subset of thewells in the plate is selected. In some embodiments, the subset includesat least one well that contains a liquid or a sample, though this is notnecessary for the method disclosed herein. For at least some wells inthe subset, and in some embodiments for all the wells in the subset, aposition of the plate bottom surface is identified with respect to afirst objective lens having a first magnification, such as the objectivelens of first scanning device 402 of FIG. 4, at step 502.

In the context of the present application, the plate bottom surface isdefined as the lower surface of the plate bottom, which interfaces withthe environment surrounding the plate, such that at the plate bottomsurface the illumination beam (laser beam) transfers from the airsurrounding the plate to the glass or plastic of the plate.

Typically, the first objective lens has a first magnification, such asfor example 10×, 20× or the like.

The position of the plate bottom surface in the wells of the subset maybe identified using any suitable method known in the art, including bothmanual and automatic methods. In some embodiments, the positions of theplate bottom surface are identified substantially as described in U.S.Pat. No. 7,109,459, which is incorporated by reference as if fully setforth herein, substantially as described hereinabove.

At step 504, the identified positions of the plate bottom surfaces atthe wells in the subset are stored in a storage unit, such as storageunit 416 of FIG. 4, together with information relating to the alignmentof the sample plate along X and Y axes within the first scanning device.For example, the alignment information may include information relatingto the X-Y position of the first objective lens relative to the plate orto each well thereof, as well as the distance between the firstobjective lens and the sample plate or a marked area thereof, or the Zposition of the first objective lens relative to the plate or to eachwell thereof, during scanning of the plate using the first objectivelens. The plate may then optionally be removed from the first scanningdevice at step 506.

At some later time, at step 508 the plate is placed in its dedicatedlocation in a second scanning device forming part of the scanningsystem, for example second scanning device 420 of FIG. 4. However, insome embodiments, the second scanning device may be the same as thefirst scanning device, and the plate may be reinserted into the firstscanning device after being removed therefrom.

The second scanning device includes a second objective lens. At step 510the plate is aligned in the second scanning device so as to be aligned,along the X and Y axes in the second scanning device, in a positioncorresponding to that used in the first scanning device, based on thealignment data stored in the storage element 416.

At step 512, using the scanning device and the second objective lens andbeginning at the position along the Z axis of the plate bottom surfaceas identified in step 502, the second scanning device scans the wells inthe subset to identify the well bottom surface of each such well.

The second objective lens has a magnification that is not greater thanthe magnification of the first objective lens. As such, in someembodiments the second magnification is smaller than the firstmagnification. In some embodiments, the second magnification may be forexample, 2× or 4×. In some embodiments, the second magnification isequal to the first magnification, but the numerical aperture value ofthe second objective lens is higher than the numerical aperture value ofthe first objective lens. In some embodiments, the ratio between thefirst magnification and the second magnification is in the range of1-10, 1-5, 1-3, or 1-2.

Based on the identified position of the well bottom surface, the wellsin the subset are scanned or imaged at step 514, using the secondobjective lens.

The scanning at step 514 may be carried out using any suitable methodknown in the art, including capturing an image stack, which isparticularly useful when imaging a three dimensional construct such as aspheroid.

In some embodiments, the second objective lens and the first objectivelens form part of a single device, so that a single imaging device isused as the first and the second imaging device. In such embodiments,the plate may be removed from the device and then may be returned to thesame device for identification of the well bottom surface. Alternatelythe steps of removing the plate from the device (step 506), placing theplate in the second device (step 508), and aligning the plate in thesecond device (step 510), may be obviated altogether.

In some embodiments, the second objective lens is an oil immersionobjective lens or a water immersion objective lens.

In some embodiments, the teachings herein may be carried out on a platehaving a single well, for example with respect to in multiple fields ofthe single well, or on a single well within a multi-well plate.

Turning now to FIG. 5B, where like steps are labeled by like referencenumerals, as seen at step 500, a subset of the wells in the plate isselected, substantially as described hereinabove with respect to FIG.5A. For at least some wells in the subset, and in some embodiments forall the wells in the subset, a position of the plate bottom surface aswell as a position of the well bottom surface is identified with respectto a first objective lens having a first magnification, such as theobjective lens of first scanning device 402 of FIG. 4, at step 502 b.

As discussed hereinabove, typically, the first objective lens has afirst magnification, such as for example 10×, 20×, 40× or the like.

The positions of the plate bottom surface and the well bottom surface inthe wells of the subset may be identified using any suitable methodknown in the art, including both manual and automatic methods. In someembodiments, the positions of the plate bottom surface are identifiedsubstantially as described in U.S. Pat. No. 7,109,459, which isincorporated by reference as if fully set forth herein, substantially asdescribed hereinabove.

At step 504 b, the identified positions of the plate bottom surfaces andwell bottom surfaces at the wells in the subset are stored in a storageunit, such as storage unit 416 of FIG. 4, together with informationrelating to the alignment of the sample plate along X and Y axes withinthe first scanning device, substantially as describe hereinabove. Theplate may then optionally be removed from the first scanning device atstep 506.

At some later time, at step 508 the plate is placed in its dedicatedlocation in a second scanning device forming part of the imaging system,for example second scanning device 420 of FIG. 4. However, in someembodiments, the second scanning device may be the same as the firstscanning device, and the plate may be reinserted into the first scanningdevice after being removed therefrom.

The second scanning device includes a second objective lens. At step 510the plate is aligned in the second scanning device so as to be aligned,along X, Y, and Z axes in the second scanning device, in a positioncorresponding to that used in the first scanning device, based on thealignment data stored in the storage element 416.

At step 512 b, based on the position of the well bottom surfaceidentified in step 502 b, the wells in the subset are scanned or imagedusing the second objective lens.

The scanning at step 512 b may be carried out using any suitable methodknown in the art, including capturing an image stack, which isparticularly useful when imaging a three dimensional construct such as aspheroid.

As discussed hereinabove, the second objective lens has a magnificationthat is not greater than the magnification of the first objective lens.As such, in some embodiments the second magnification is smaller thanthe first magnification. In some embodiments, the second magnificationmay be for example, 2× or 4×. In some embodiments, the secondmagnification is equal to the first magnification, but the numericalaperture value of the second objective lens is higher than the numericalaperture value of the first objective lens. In some embodiments, theratio between the first magnification and the second magnification is inthe range of 1-10, 1-5, 1-3, or 1-2.

In some embodiments, at step 516, prior to the scanning step 512 b, acorresponding in-focus position for the well bottom surface with respectto the second objective lens is computed based on the information storedat step 504. In some embodiments, the computation of the correspondingin-focus position is based also on the difference in magnificationand/or in numerical aperture between the first and second objectivelenses. In some embodiments, the second objective lens is an oilimmersion objective lens or a water immersion objective lens, and thecomputation of a corresponding in-focus position is based (also) on thedifferences in optical characteristics between a standard (air)objective lens and an oil immersion objective lens or water immersionobjective lens.

In some embodiments, the second objective lens and the first objectivelens form part of a single device, so that a single scanning device isused as the first and the second scanning device. In such embodiments,the plate may be removed from the device and then may be returned to thesame device for identification of the well bottom surface. Alternatelythe steps of removing the plate from the device (step 506), placing theplate in the second device (step 508), and aligning the plate in thesecond device (step 510), may be obviated altogether.

In some embodiments, the teachings herein may be carried out on a platehaving a single well, for example with respect to in multiple fields ofthe single well, or on a single well within a multi-well plate.

It will be appreciated that the teachings herein allow the scanningdevice to be in-focus with respect to the plate regardless of the“expected height difference” and of the “unexpected height difference”within the plate. The “expected height difference” is defined as thecurvature of the plate listed in the specifications of the plate andthat is intended by the manufacturer to be in the plate, such as havinga curved bottom due to the structure. The “unexpected height difference”is defined as lack of planarity which is not intended in thespecification of the plate. Such “unexpected height difference” may be,for example, due to differences in the relative heights of the bottomsof the wells; or may be, for example, due to deviations from planarityin the virtual surface traced by the scanning components as theobjective is moved; or the surface upon which the plate rests beingnon-parallel with the virtual surface traced by the scanning componentsas the objective is moved.

It will be appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the scope of the appendedclaims.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

The invention claimed is:
 1. A method for scanning at least some wellsof a plurality of wells in at least a portion of a multi-well plate, themethod comprising: using a first objective lens having a firstmagnification to identify, in each of at least three wells of a selectedsubset of said plurality of wells, an in-focus position of each of saidat least three wells with respect to said first objective lens; using atleast three said in-focus positions as a basis to compute a plane alongwhich said at least three wells will be in focus with respect to atleast one objective lens having a second magnification that is notgreater than said first magnification; and using said at least oneobjective lens to scan, along said plane, at least some of saidplurality of wells in said portion of said plate.
 2. The method of claim1, wherein said at least one objective lens is said first objectivelens, and said first magnification is equal to said secondmagnification.
 3. The method of claim 1, wherein said at least oneobjective lens is a second objective lens, different from said firstobjective lens, wherein said second magnification is smaller than saidfirst magnification.
 4. The method of claim 1, wherein said scanningusing said at least one objective lens is carried out without carryingout additional focusing operations.
 5. The method of claim 1, whereineach of said wells comprises a generally cylindrical side wall, and abottom surface comprising a portion of at least one of a sphere, aparaboloid, and an ellipsoid.
 6. The method of claim 1, wherein each ofsaid wells comprises a generally cylindrical side wall, and a planarbottom surface.
 7. The method of claim 1, wherein each of said wells isfrusto-conical.
 8. The method of claim 1, wherein said using a firstobjective lens is carried out in a first scanning device and said usingsaid at least one objective lens is carried out in a second scanningdevice, and wherein the method further comprises, following computationof said plane and prior to said using said at least one objective lens:storing alignment data of said plate along X and Y axes of said plate insaid first scanning device, and data relating to said plane, in acomputer storage element which is in communication with said first andsaid second scanning devices; moving said multi-well plate to saidsecond scanning device; and aligning said multi-well plate along said Xand Y axes of said plate in said second scanning device based on saidalignment data stored in said computer storage element.
 9. A method forscanning at least a portion of a well in a plate, the method comprising:using a first objective lens having a first magnification to identify,at at least one location of said well, a first in-focus position of atleast a portion of said well with respect to said first objective lens;identifying, for said first in-focus position, a corresponding in-focusposition with respect to a second objective lens, different from saidfirst objective lens and having a second magnification, based on opticalcharacteristics of said at least one objective lens; and using said atleast one objective lens to scan, at a height corresponding to saidcorresponding in-focus position, at least said portion of said well,wherein said second magnification is smaller than said firstmagnification.
 10. The method of claim 9, wherein said scanning usingsaid at least one objective is carried out without carrying outadditional focusing operations.
 11. The method of claim 9, wherein saidwell comprises generally cylindrical side walls, and a bottom surfacecomprising a portion of at least one of a sphere, a paraboloid, and anellipsoid.
 12. The method of claim 9, wherein said well isfrusto-conical.
 13. The method of claim 9, wherein said using a firstobjective lens is carried out in a first scanning device and said usingsaid at least one objective lens is carried out in a second scanningdevice, and wherein the method further comprises, following identifyingsaid corresponding in-focus position and prior to said using said atleast one objective lens: storing alignment data relating to alignmentof said plate along X and Y axes of said plate in said first scanningdevice, and data relating to said corresponding in focus position, in acomputer storage element which is in communication with said first andsaid second scanning devices; moving said plate to said second scanningdevice; and aligning said well in said plate in said second scanningdevice based on said alignment data stored in said computer storageelement.
 14. An auto-focusing device for automatically determining anin-focus position of a plurality of wells located in at least a portionof a plate containing wells, the device comprising: a computationcomponent programmed to compute a plane along which at least three wellsin said portion of said plate would be in focus with respect to anobjective lens; a first objective lens functionally associated with saidcomputation component, said first objective lens having a firstmagnification, signals obtained with said first objective lens beingused by said computation component for identifying an in-focus positionfor each of at least three wells of a selected subset of said pluralityof wells; and at least one objective lens having a second magnification,said second magnification being not greater than said firstmagnification, for scanning at least some of said plurality of wells insaid portion of said plate along said plane, wherein said computationcomponent is configured to compute said plane along which said at leastthree wells would be in-focus with respect to said at least oneobjective lens based on at least three said in-focus positions.
 15. Theauto-focusing device of claim 14, wherein said at least one objectivelens is configured to scan said plurality of wells along said planewithout carrying out additional focusing operations.
 16. Theauto-focusing device of claim 14, wherein said at least one objectivelens is said first objective lens, said second magnification is equal tosaid first magnification.
 17. The auto-focusing device of claim 14,wherein said at least one objective lens is a second objective lens,different from said first objective lens, and wherein said secondmagnification is smaller than said first magnification.
 18. Theauto-focusing device of claim 14, wherein the device is configured foruse with a plate in which each of said wells comprises a generallycylindrical side wall, and a bottom surface comprising at least one of aportion of a sphere, a paraboloid, and an ellipsoid.
 19. Theauto-focusing device of claim 14, wherein the device is configured foruse with a plate in which each of said wells is frusto-conical.